Cell Lines with a Single Functional Isoform of the IP₃ Receptor

Abstract

Many agonists regulate cellular functions through surface receptors coupled by the phosphoinositide cascade to Са2+ mobilization. In unexcitable cells, the generation of Са2+ signals relies largely on Са2+ release from из Са2+ store localized in the endoplasmic reticulum (ER). In this system, IP3 receptors (IP3Rs), which are intracellular IP3-gated Са2+- channels, mediate controllable Ca2+ release from Ca2+ store in response to cell stimulation. A variety of factors embarrass both the analysis of a specific role for IP3Rs in cell physiology and detailing their regulatory mechanisms. Firstly, three genes encode IP3Rs, while cells usually express two or even all of them. Moreover, different IP3R isoforms are under control of distinct mechanisms. Yet, isoform-specific antagonists of IP3Rs have not been identified yet. Cell lines that express a single isoform appear to be an effective cellular model for studying regulatory mechanisms, pharmacology, and physiological role of I-P3R subtypes. Here we employed the CRISPR/Cas9 technology to inactivate genes of IP3Rs in cells of the HEK-293 line, which express all three isoforms. Several monoclones of genetically modified HEK-293 were obtained, and those containing biallelic inactivation mutation in two IP3R genes were identified. Finally, three monoclonal cell lines, each containing the only functional IP3R isoform, were generated. These cellular models can be used to evaluate a role for IP3R of a particular subtype in agonist-induced Ca2+ signaling as well as for the analysis of their regulatory mechanisms.